rare homologous recombinant clones - (Apr/16/2008 )

Hi

i am new to gene targeting. I experience very low rate of homologous recombinant clones to the surviving clones ~1:500. Does any one have any suggestion to increase the frequency?

Hi,

maybe you should tell us a bit more about your experiment.

Which cells did you use?
How did you do it?
What does your targeting construct look like?
How do you select for the recombinant clones?

Vista

Hi, vista,
EmbryoMax® Embryonic Stem Cell Line - Strain 129/SVEV. I am using a clone that is already has one loxP and I want to add another loxP upstream by about 75kb. DNA introduction is by electroporation. About the vector: LA(7.5kb)-LoxP-PGKneo-LoxP(1.4kb)-RA(1.5kb), selection: +ve G418 , -ve DT.

Hi,

Are you sure, that your selection works well? Do you use untransfected cells as a control and do all of them die? (we had the problem, that the concentration was too low, but we didn't realize it as the the control was discarded too early)
The DT-selection will not kill all the "wrong" clones, it will only decrease the rate by about 10%.
Does your electroporation work well?

Your homologous arms sound a bit short to me, but I do not work with stem cells and I use a vector based on promotor trap - it might be different there.